The ability to genetically modify donor cells and using them for nuclear transfer provides a tool for the production of genetically modified animals which may be used for example as disease models for the study of serious human diseases and drug testing.
Traditional cell nuclear transfer techniques involve two steps of micromanipulation. A first step involves the enucleation of a mature oocyte, and a second step encompasses the transfer of a donor nucleus. Micromanipulation, however, has proven to have several disadvantages for example the need for expensive equipment, the need for highly skilled personnel and time-consuming work.
An improved method of nuclear transfer employing somatic cells as donor cells has been developed recently, a method known as Hand-Made Cloning (HMC) which involves the use of zona pellucida free oocytes. The method is simplified in comparison with the traditional nuclear transfer as micromanipulation is no longer needed. The method has been used in bovine (Vajta et al. 2001 Cloning 3, 89-95; Vajta et al. 2003 Biol. Reprod. 68, 571-578; Vajta et al. 2005 Reprod, Fertil. Dev. 17, 1-16; Tecirlioglu, et al., 2004). Also the use of zona-free nuclear transfer with one step of micromanipulation has been described for bovine (Booth et al. 2001 Cloning Stem Cells 3, 139-150; Oback et al. Cloning Stem Cells 5, 3-12) and porcine (Booth et al. 2001 Cloning Stem Cells 3, 191-197). The fact that this method is technically less demanding and less time-consuming has prompted researchers to suggest applying the HMC technique to other species. However, a number of technical problems made HMC application in pig more demanding than originally supposed. One of the problems encountered relates to low buoyant densities of porcine oocytes, both Zona intact (ZI) and especially zona-free (ZF) porcine oocytes. Consequently, porcine oocytes do not settle to the bottom of the dish. Furthermore, the surface of the oocytes is sticky and it is hard to avoid their attachment to each other when zona is removed. Moreover, ZF porcine oocytes are very fragile and it is difficult to bisect them in the way as described for bovine oocytes.
Recently, the HMC technique was, however with low efficiency, applied in porcine nuclear transfer, using genetically modified somatic cells, fibroblasts, as donor cells resulting in the production of genetically modified cloned blastocysts (Kragh et al. 2004 Reproduction, Fertility and Development 16, 315-318).
The present invention improves the technique for somatic cell nuclear transfer through HMC resulting in an increased embryo reconstruction rate and consequently the chance of obtaining genetically modified animals is increased significantly.
An obstacle to producing genetically modified animals by nuclear transfer methods at a large scale is the inability of cryopreserving pig oocytes and embryos using methods applied to other species. This is due to a high lipid content of porcine oocytes and embryos. Cryopreservation of cloned porcine embryos may considerably improve the output of somatic cell cloning by alleviating logistic problems. However, recently a noninvasive procedure was published for delipation of porcine embryos with centrifugation but without subsequent micromanipulation (Esaki et al. 2004 Biol Reprod. 71, 432-6).